Therapeutically active aromatic diazo compounds and derivatives thereof



Patented Nov. o, 1945 a V v 2,388,260

UNITED STATES, PATENT OFFICE" THERAPEUTICALLY lic'rivn nnom'rlo nmzo COMPOUNDS AND nnmvs'rrviis THEREOF most A. n. Friedheim, New York, N. x.

No Drawing. Application March 11, 1942,

Serial No. 434,176

7 Claims. (Cl- 167-48) This invention relates to new condensation nous. high molecular materials extraneous to the products formed by a reaction between aromatic human organism; nd capable P y pa diazo com ounds, which may be known products as p hogenic agents inhuman patholo or compounds or ,a new type described herein- The diszo mp u su l for rryins out after, and the nitrogenous materials s eeined a this i e t a p u d w ic con ai r hereinafter, and to the method of carrying out are capable of li r in a diazo group f the said reaction between said diazo com ounds and iollowins rmu said nitrogenous materials. It has particular (1) relation to the condensation of said diazo compounds with natural, high molecular nitrogenous 10 materials extraneous to the human organism, group consisting of saturated or unsaturated which are capable of playing a part as a pathomonoor polynuclear, isocyclic or heterocyclic genie agent, nd may be isolated products or structures such as benzene, naphthalene, tetraingredients of substances of animal or vegetable hydrqnaphthalene, diphenyl, anthracene, Py origin, or living microorganisms or their compo- 1o di qu olin ra icals. nd A maybe any subnents-said condensation products being new stitu'ent except OH and a substituent containing products having no or reduced pathogenic eflect fl'ee mono-Substituted amino P- m i in comparison with the nitrogenous substances dlcates the number of A radicals. w n 1 used as starting materials. the A-radicals may be the same or. different.

wherein Ar represents a radical selected from the I have made-the surprising observation that (Na) denotes a diam-group, and m the number v diazo compounds of the structure specified hereof diazo-groups. The Ar radical may be, for inaiter, which generally are colorless or slightly example, substituted by one or more hydrogen, colored only, condense readily at neutral or alhalogen, alkyl, O-alkyl, N-dialkyl, N02, CONHa,

kaline reaction with nitrogenous materials of the COOH, SOsH, AsOsHz and groups of the formula above described type to form colored condenser- 802.13. In this formula, E represents an amide tion products. Said materials comprise the radical selected irom the group consisting of pathogenic nitrogen bearing materials as exist imides, such'as guanldine and substituted guaniin the form of poisons derived from cold blooded dines, and amine radicals, such as Ni -I2, and animals such as reptiles, i. e. snakes, fish, ammonoor, disubstituted NH: groups substituted by phibia, i. e. frogs, arthropodni. e. scorpions, spi- 3o aliphatic or aromatic, components. The cyclic ders, mosquitos, in the form of bodies, and body components may be isoor heterocyclic, monofractions of living and dead microorganisms, said r ciynuelear, and saturated or nonsaturated. microorganisms including protozoae, such as Thus, E may represent a NH: group which may fia l 'f. e. tr p no omes, rhiz pods. fe. besubstituted, for example, by benzene and hoys nt ric m ba haematoloa, p fl 5 mologous radicals, pyridine, pyrimidine, thlazole, dium malariae, lower forms of life, such as lower n t iazin radicals, fungi, if. e. trychophyton, bacteria, bacilli, cocci, 1 have found th t th se of acid. substituents, pi h r ket ia, virus, inthe f rm f p such as COOK and 503K, in theAr radical is ucts produced by, or under the action of said particularly advantageous because such acid radmicroorganisms such as endoand ectotoxins, in 40 icals s re gobd solubility and the diazo comthe form of so-called allergenic substances or pounds containing them, yield condensation r ns der ved fr m animals, plants. and'lowroducts of, a particularly high degree of noner forms of life including p l n allergens and toxicity. The above mentioned substituents of tuberculin. The resulting condensation products "the Ar radical are merely illustrative, and a great are harmless or of reduced harmfulness to human 5 number of other substituents may be used as Organisms- My invention may also be carri d be understood by those skilled in the art. out with or in organisms containing such nitrog- According t on referred embodiment of my enous substances, for example, with living or invention, 1 use diazo compounds, in which h dead bodies of microorganisms or fractions therediazo radicals of the above mentioned group of. The invention is operative if said bodies of 1 v i microorgwisms are located in mm or m are connected with organic acid radicals having such as on infected normal or pathological surthe 1 ma faces of the animal The components [Du Ara-A, 1 (n) which react with the diazo compound according j sn q t my invention, are always n tur nitr s wherein Ar and A are radicals selected from I nitrogenous material.

2 2,888,260 v the same group as Ar and A and may be the same volition. It the as Ar and A, respectively, or diflerent, and in which D is an acid radical selected from the group formation or my con consisting of COOH and 803K. In the simple g oi most of th ensuing compound has the eneral formula [Aa Ar (Nz)]n .[Da Ar'-A'n (m) where 115 may be equal to us or smaller, but at least one.

the general formula |:A1| A1"(N'2)1| ]u .[1?n A1" 5: 11 (IV) Every diazo radical (N2) must'be saturated by radical is formed These compounds salts, wherein the tem carrying the diazo group or groups is substituted by at least the same number of acid radicals as diazo groups according to the general formula wherein 11. .1 and preferably n and n: are the same and equal to 1 or 2 Such preferred compounds are p-diazo-benzene-suli'onic acid of the formula SOs-CrHc-Na =3.3'=l-' enzidine dlsulfonic acid of the formula Ive-(camera the following schemes written for the simplest case:

( I) [A-AI-Nz] -oom und a Diazogroup [N m I High molecular compound v stream-ra The inner salts of the above Formula v are hydrolyzed in contact with water to form amphoteric ions of the formula I have found that the above mentioned toxic products are rendered non-pathogenic or considerably less pathogenic'if subjected to condensation with diazo compounds according to my indiam compounds are caused to react with living forms of microorganisms. the

densation products results in e living forms of said micro- 5 organisms, as the condensation introduces foreign radicals into case their known influences.

high molecular their body and acid fast microorganisms show higher resistance It has been found that the introduction or a maximum amount oi N=N substance.

to chemical links into the nitrogenous material results in nitrogen atom present in the reacting high molecular I ha

cases particularly valuable condensation prodalso in this surfaces of'an animal with the surface 'ture with a salt bufler such diluent, I prefer rying the diazo radical ,dlazo derivatives of such them as local antiseptics,

aseasoo and reaction with .the pathogenic material. Otherwise the necessary amount of moisture may be applied, in the form of water or preferably a physiological salt solution. Applied to infected body, the diazo compounds react readily with the infecting living micro scribed,andwhile such solutions organisms, their high molecular, nitrogen'ous' toxic, metaand catabolic products, as well as of the tissues. While most species of living microorganisms are killed by this reaction, the host cells are substantially not damaged but only colored on their surface.

If the reaction between the diazo compound is to be carried out on surfaces of the animal body,

such as 'in wounds, a physiological pH of the aqueous reaction mixture should be assured. According to this embodimentof myinvention, I may use, therefore, the diazo compounds in mixselected, from the group consisting of primary and secondary alkali phosphates, and primary and secondary alkali carbonates. Furthermore, dosage may call in such cases for'the use of a diluent which isnontoxic and nonirritant, readily resorbed from the body surfaces, and, of

course, does not react with diazo radicals. As

to use a neutral alkali salt of an organic acid radical (D-Ar').

The use of an alkali carbonate as a salt buffer in mixture with a compound carrying at least one free acid radical, or an acid radical capable of being set free by hydrolysis, results in the advantageous effect of liberating CO2. On applying, for example, such a mixture to the moist surface of a wound, gaseous CO2 is set free,which forms a foam with the secretions of the wound, thus contributing to the mechanical cleansing of the wound, as the foam carries extraneous parof the formulaholes, which may be present in the depth of the wound, to the surface.

The above mentioned be present in the molecule caror in the diluent (see Ex- If the molecule carryacid radical may amples VII and VIII).

- ing the diazo radical is a salt, the acid radical may be a substituent of Ar or In carrying out my present invention by the application of a diazo compound to .the animal.

body, I have found that a particular advantage may be obtained by the use of diazo derivatives of aromatic amines known to have a particular chemo-therapeutic effect, such and the products derived from the same by substitution in the SOzNHr group. By applying aromatic amines toa wound of the human body, molecular, nitrogenous organic substances of microorganisms present in the wound, and their metaand catabolic products,-and the diazo derivatives of said aromatic amines form detoxi fled condensation products which may be converted in the course of time within the human organism with liberation of free sulfonamides which, may then exert their specific chemotherapeutic eflect in statu nascendi.

- Thus, in carrying out this embodiment of my present invention, the diazo compounds of said aromatic amines have an and intheir use, the new antiseptic and. detoxifying effect obtained of free sulfonamides.

The compounds used invention are sulfonamide in this embodiment of my derivatives of diazorequirements of medical I effect which classes benzene. While the treatment of acid solutions of sulfanilamides with nitrous acid has been dehave been used for the preparation of azo dyestuiis, and asides, free diazo bases, and especially stable dlaao compounds'of benzene sulfonamides and their substitution products have not been isolated or prepared, respectively, in dry solid form prior to my invention. I have prepared these compounds according to the method described in inafter.

The above mentioned sulfonamide derivatives of diazobenzene have the general formula stituent containing a free or mono substituted A acid, n is equal wherein x is selected from the group of OED-alkali, inorganic acid groups, cals of aliphatic or acids, said radicals having no substituent selected from the group consisting of OH and a suband radiamino group. n being equal to the number of diazobe'nzene sulfonamide groups connected'to X. and Y is anitrogenous ondary or preferablyprimar amino and imino radicals. If .X is the radical of a dior polybasic to or smaller than the valency of said acid, but at least one. As to the nature of taking the place of x, satacids may be used. Benzeneand naphthalene monoandpolysulfonic acids answer all practical purposes. In view of their ready availability, benzene mono and disulfonic acid, naphthalene=l,5=disulfonic acid, naphthalene==l,3 ,6= trisulfonic acid, and naphthalene, 1,3,6,8 -te trasulfonic acid are preferred. Further examples are: Toluene mono-, diand trisulfonic acid, and

. tetrahydronaphthalene disulfonic acid. The cyeffect of these diazobenzene,

"ing to my invention clic sulfonic acids may be substituted by one or more halogen, alkyl, N-alkyl, N-diaikyl, N02, COOH, CONHa, SOME: radicals, for. example. The new primary bactericidal and detoxifying derivatives accordis not substantially influenced by the nitrogenous radical taking the place of Y in the above general formula, as said effect is a.function of the diazo radical in the condensation with high molecular, nitrogenous compounds.

as sulfanilamide t for example, the high I combined with compounds, the .benzene- As examples of the radicals taking Y, the following may be mentioned: alkyl, N-dialkyl, NH acyl,

the place of NH-4611s) n-NH2, Nmcmm- OHOI-I, NH (cm) n-CON'H:

-NH.C.NH| isocyclic nitrogenous radicals, such as aniline, naphthylamine and their substitution products.

I prefer to use such nitrogenous radicals which produce a specific chemo-therapeutic effect when the NHs-CsH4-SOz=radical such as amino derivatives selected from the group consisting of amino-pyridine, amino pyrimidine,

amino-thiazole and guanidine and substituted As preferred examples of suitable and naphthalenemono-, diand polysulfonates of p-diazo-benzene-sulfonamido-pyridine (diazo sulfapyridine) p-diazo-benzene-sulfonamido pyrimidine (diazo guanidines,

sulfadiazine); p-diam-benzene-sulfonamido-thiazole (diazo 'sulfathiazole) p-diazo-benzene-sulfonyl-guanidine (diazo sulfaguanidine) and p-didetail here- 7 consisting preferably cyclic sulfonic radical selected fromthe group consisting of aliphatic and aromatic secand polynuciear, and heterocyclic azo benzene-sulfonyleacetyl-imide (diazo sulfamyd) may be mentioned. Sulfonaanide derivatives of diazo-benzene, in which a sulfonic acid from which low molecular benzene sulfonamide derivatives are set free within the organism.

Example I.100 cc. of a diphtheria toxin obtained and purified according to known methods, and corresponding to 20 LF units per cc., are cooled to C., and caused to react under stirring with 3 cc. of a 1% neutralized, aqueous solution of freshly recrystallized p-diazobenzene sulfonic acid. The reaction mixture, which takes up an orange-red color, is kept at about 8 C. for about 12 hours. After this period of time the free'diazo compound has disappeared in the solution, and formation Of a condensation product from the toxin and the diazobenzene sulfonic acid is completed. Tests made with this solution in experiments on animals show that the reaction product is 800,000 times less toxic than the toxin used as starting material, while flocculation test shows that the specific antigen eflect of binding and fiocculating diphtheria serum is not decreased in comparison with the original toxin used.

Example. 1!.1 g. of dried poison of Lachesis alternatus is dissolved in cc. of a phosphate buiIer solution moi/15, pH 7.3. To this ice-cold solution 33 cc. of a 1% solution of p-diazobenzene sulfonic acid are added drop by drop under stirring. The reaction mixture, which takes up an orange-red color almost instantaneously, is kept at about 5 C. for about 24 hours, after which period no free diazo compound can be found in r the solution. A dose of 200 mat/kg. of the condensation product thus formed can be administered without adverse eflect to mice by intravenous injection, while a dose of 0.75 mg./kg. of the original poison used here as starting material, is lethal to mice.

Example [IL-l0 cc. of an ice-cold solution of fonic acid are added under shaking. The crystals go into solutio and the reaction mixture takes up a reddish-brown color. After keeping this reaction mixture at about 8 C. for about 15 hours, no free diazo compound can be found in the solution. If the untreated starting material here used is administered by intracutaneous iniectionto an allergic patient in a dose corresponding to 0.000001 g. of nitrogen, it causes a strong reaction, while the condensation product obtained according to this example can be administered, without causing a reaction, in an 8,000 times higher dose.

Example V.- cc. of a 1% lobster extract obtained according to known methods and butfered to pH 7.6, is gradually mixed under stirring with 0.25 g. of crystallized diazobenzene sulfonic acid. The reaction mixture, which takes up a reddish-brown color, is kept at about 8 C.-

for about 48 hours, after which period no free diazo compound is present in the solution. If the original extract here used is administered by intracutaneous injection in a dose corresponding to 0.0000001 g. of nitrogen to an allergic patient, it causes a distinct reaction (erythema), while a 10,000 times higher dose of the product here obtained can be administered under substantially similar conditions without causing a reaction.

Example VI.--100 cc. oi a suspension of virulent typhus bacilli in physiological sodium chloride solution containing 5.10 bacilli per cc.. and obtained by dispersing a 24 hours old culture of typhus bacilli on nutritive agar, are cooled to 2 C.,'and mixed under stirring with 10 cc. of a 1% neutralized solution of p-diazobenzene sulfonic acid. Upon mixing said suspension with said solution, the suspended bacilli take up a brownish-red color almost instantaneously, and inoculation tests on bouillon show that the suspension is sterilized within l5 minutes. The colpurified tuberculin, prepared according to Seibert (Am. Rev. of Tuberculosis, Suppl. 1934, 30, .p.

707), in a /15 mol phosphate bufler solution, pH 7.4, are gradually mixed under stirring with 7.2 mg. of freshly recrystallized p-diazobenzene sulfonic acid. The reaction mixture, which takes up an orange-red color, is kept at about 5 C. for about 12 hours, after which period no free diazo compound can be detected in the solution. While intracutaneous injection to tuberculous guinea pigs of 10" g. of the original tuberculin,

here used causes adistinct reaction (erythema of 15 mm. diameter), the reaction product prepared according to this example can be injected under substantially similar conditions to guinea pigs in a 10,000 times higher dose without causing a reaction.

Example IV.-To an ice-cold aqueous solution of pH 7.6, containing 1% of the extractive substances of the water-soluble fraction obtained according to known methods from Artemisa tenuifolia, 35 mg. of crystallized p-diazobenzene sulored bodies of bacteria are then centriiuged and used for immunizing guinea pigs. A serum obtained after 14 days from the latter agglutinates untreated typhus bacilli with 8 titer 1:300. While 1 cc. of the original suspension sterilized by heat, causes severest symptoms when intraperitoneally administered to guinea pigs, a five times higher number of the bacilli condensed with the diazo compoundas described above, can be applied under similar conditions without causing any symptoms.

In the above examples, p-diazobenzene sulfonic acid may be substituted by any other compound of theabove Formula I or preferably III.

Example VIL-A finely pulverized mixture is prepared by mixing equimolecular parts of diazobenzene sulfonic acid, sodium carbonate, and sodium benzenesulfonate. This mixture is applied by dusting it on wounds either for prophylactic or therapeutic purposes.

Example VIII.-One part by weight of benzenesulfonate of diazobenzene sulionamide, and 2 parts by weight of an equimolecular mixture of toluene sulfonic acid and sodium carbonate, are intimately mixed and applied to wounds in solid form by dusting. Instead of the benzenesulfonate of diazobenzene sulfonamide, one part by weight of the corresponding toluene-sulfonate may be used.

In order to prepare the benzene-sulfonate of diazo-benzene sulfonarnide, a solution of 17.6 g.

y 'with eilicient stirring,

7 dissolved in 30 cc. oi water, are added to the rein Example assaaeo dissolved in 36 cc. of

6.9 g. of sodium nitrite water. n addition of 15.8 g. oibenzene-sulionic acid to the yellowish solutioma white crystailine precipitate oi benzenesulionate'oi diazois'filtered ice-water, alcohol and ether. compound is soluble in waand hydrochloric acid. The

colorless aqueous solution yields on addition oi sodium acetate or sodium bicarbonate an amorphous, yellow precipitate oi iree diazobenzenesulionamide. The aqueous solution is neutral and couples with R iormred azo-dyes.

The compound may be recrystallized out of warm water. It stands heating up to 110 C., while at higher temperatures it becomes soft and discolored, and is decomposed at about 128 C.

In order to prepare the toluenesulionate of diazobenzene sulionic acid, 1 part by weight oi diazobenzene sulionate and 1.1 parts by weight of toluene-sulionic acid are suspended in 8 parts of water. The suspension is stirred for 1 hour at 25 C. and kept for 12 hours at 5 C. The white precipitate oi toluenesulionate oi diazobenzene sulionic acid is then filtered oil, and washed with ice-water, ethylalcohol and ether.

, The compound can be saiely dried at 80 C.

Example IX.Equal parts or solid, crystallized diazobenzene sulionic acid; prepared according to classical method, and benzene sulionic acid are dissolved in 20 parts of warm water of about 8}) C. On cooling, a white crystalline precipitate oi benzenesulionate oi diazobenzene suiionic acid is obtained, which is filtered oil, and washed with acetone and ether.

The product thus prepared may be used in an equimolecular mixture with sodium carbonate and an. appropriate diluent such as disodium naphthalene disulionate.

Example X.-20 cc. of molten benzenesulionic acid are added to a solution oi 257 g. of suliathiazole in 450 cc. of water,'and 25 cc. of hydrochloric acid (D=1.19) A white precipitate of suliathiazole benzenesulionate is iormed. Regardless oi this precipitate, 300 g. oi ice, and, 7.02 g. of sodium nitrite action mixture. Stirring is continued untilall oi the white suliathiazole benzenesulionate is replaced by a yellow precipitate consisting oi 'diazothiazoie benzenesulionate which is filteredoii, washed with ice-water, acetone and ether. The yield is 37 g., i. e.

The compound thus obtained is soluble in wa-. ter, and couples with R salt to form an azo dye. It may beused ior iorming condensation products according to my invention, without any admixture or in a mixture similar to that described 12, by dusting it on wounds. Example XL-A solution oi 29 got suliapyridine in 250 cc. oi water and 40 cc. of hydrochloric acid (D=l.l9), and 200 g. or ice is diazotized by the addition of 7.0 g. of sodium nitrite dissolved in 35 cc. of water. Diazosuliapyridine precipitates in tom of fine, yellow needles which are filtered off, washed with a small amount of ice-water, and redissolved in 300 cc. oi methanol. To the filtered alcoholic solution, 3'1 cc. oi molten benzenesulionic acid are added with good shak- 70,

in: and cooling. A yellow, crystalline precipitate oi diazosuliapyridine benzenesulionate is formed, which is filtered oil, and washed with methanol and ether. The yield amounts to 25 g., i. e. 64% oi the theoretical value.

salt and naphthylamine to suiionate is not stable-to about 130 C.

the latter couples readily with an alkaline soluexample,

,disulionic acid, andi mol is applied to wounds by dusting, with the addi- The compound to a clear solution standing. whllethe acetone and dioxane. While the free diazonium base, in solid. dry form, mg to about wt-80 C.,

An aqueous solution oi mm of R salt, and an acid solution of naphthylamine to term red azo dyes.

The diazosuliapyridine benzene sulionate may be reacted according to my present invention with high molecular, nitrogenous compounds, for

in the manner Example XII.--One part by weight oi a mixture consisting oi 1 mol oi (4,3)-4',3'-tetrazodiphenyl-disulionic acid, and

carbonate, is mixed with an equal part oi a diluent such as sodium benzene sulionate. The mixture may be applied to wounds by dusting.

Example XllL-One pm by weight of a mix-- ture consisting oi lmol 1,4,8 diazonaphthaleneoi sodium carbonate,

- tion oi a diluent ii desired.

I a finely pulverized, homogeneous Example XIV .--0ne mol each of the acid salt oi naphthalene-1,5, disulionic acid and diazosuliapyridine is mixed with 2 mols of sodium bicarbonate, and equal partsby weight of this mixhue and trisodium-naphthalene-trisulionate to Product which may be applied to infected suriace of theliving human body by dusting. The trisodium -naphthalene-trisulionrte which acts as a diluent, may

- be used in a. greater proportion or may be partly or entirely omitted.

The diazo compound. here used may be ob tained by preparing diazosuliapyridine in the manner described in Example Xi, and reacting an alcoholic solution oi this compound withan equimolecular quantity oi naphthalene-1,5-disulionic acid. Example XV.-One part by weight of an equimolecular mixture oi p-diazobenzoic acid (Na-CeHw-COU) 7' l and sodium carbonate, is mixed ,with 2 by weight oi neutral sodium benzene disulionate as diluent. The amount of the latter may bQvaried in accordance with the requirements oi dosage.

The above examples illustrate the great number of possible embodiments oi my present invention.

While in the combating oi pathological phenomena caused by pathogenic materials, partic ularly those caused by bacteria, various azo compounds have been suggested and used, such as a great number oi the so-called azo dyestuffs con- "taining an Nrgroup (-N=N) between aromatic components derived from coal tar, and hav-' .ing a pronounced bright color of their own, I have 1 discovered that another class of Nz-compounds, i. e. diazo compounds. oi the type here disclosed, whichgenerally are colorless or slightly colored only, may give even better results in combating pathological phenomena, and allow of a. much wideruse, or, in other words, allow of rendering harmless 'or less harmful a much greater number oi pathogenic materials.

As shown by the above description and examples; the pathogenic materials," with which the diazo compounds may be reacted according to ily soluble in ethanol and methanol, its benzene-5 j soluble in these solvents nor in is decomposed on heatits benzenesniionate is described in Example 31..

two mols oi sodium my present invention to form new condensation products, which are nontoxic or less toxic than the untreated pathogenic materials, include broadly high molecular, nitrogenou substances as exist in the form of poisons derived from coldblooded animals. or in the form'of microorganlame, and toxic products produced by or under the action of said microorganisms, and also in the form of the so-called allergenic substances or allergens derived from animals or plants or from lower forms of life.

It will be understood that the process of carrying the condensation according to my invention into effect, and also the specific composition of the diazo compound used. must be varied within the broad limits of my invention, depending primarily onthev nature of the high molecular nitrogen-bearing pathogenic material. For example, it is well known, that diseases caused by pathogenic materials can be combated byeither prophylactic or therapeutic treatment. Both of these methods of treatment may be supported or brought about by following the course of my invention. In the production of vaccine, I treat a toxin or a living microorganism in vitro with a diazo compound, and use the condensation prod-= uct formed for prevention of infectious diseases. Similar steps may be taken in combating other pathological phenomena such as allergic diseases. A direct therapeutic effect brought about by the destruction of pathogenic material in vivo, is obtained, for example by applying a diazo compound according to this invention to an infected wound.

Applying this invention to pathogenic substances derived from lower forms of life, which cannot be isolated in a substantially pure form, as is possible-with bacterial suspensions, in applying the invention, for example, in the case of virus diseases, such as poliomyelitis, or in ricketsia diseases, such as typhus, the condensation according to my invention may be carried out by reacting the diazo compound with a suitable pathogenic material, such as finely divided spinal cord of monkeys infected with poliomyelitis or spleen pulp of guinea pigs infected with typhus exanthematicus or cultur media containing the virus or ricketsia.

In order to destroy microorganisms, particularly bacteria, and to combat the substances produced by' and/or under the action of the same, in a living animal body, I cause said microorganisms, and/or said substances, to react with a suitable diazo compound. This can be effected, as already explained above in detail, by bringing said diazo compounds in contact with said microorganisms and substances, wherever they are present, such as in infected wounds or on other infected surfaces of the living animal body, or in vitro. Said diazo compounds may alsobe used by direct application in the treatment of mou nose, eyes, sinuses and the urogenital system. In this embodiment of my invention, the above described solid mixtures comprising diazo compounds, builer substances and/or diluents, proved to be of particular value.

The above described sulfonamide derivatives of diazobenzene are new compounds combining in a new way two eflects. First, the new detoxifying and bactericidal eflect, which I call a primary and which is based on the presence of the group and its property to link up with the high molecular nitrogenous materials, and sectypical therapeutic efiect due to sulfonend, the amides which are liberated within the organism after parenteral administration such as intra- 75 venous iniection or local application to infected surfaces.

The new sulfonamide derivatives of diazobenzene according to my invention, may be prepared according to the following general methods.

A sulfonilamide compound is diazotized in the usual way in acid aqueous solution, and the diazo compound thus formed is then reacted with an organic acid of the general formula D-Ar'. This can be done in solution or in suspension either after isolation of the diazo compound in form of the free diazo base or in form of an inorganic salt thereof, or by reacting the diazo compound dissolved or suspended in the original reaction medium. Finally, the sulfanilamide compound may be diazotized in the presence of the organic acid.

It is to be understood that in the present specification and claims, the term "bufler salt" denotes a salt which is used for maintaining a desired pH value, and is preferably selected from primary and secondary sodium phosphates and primary and secondary sodium carbonates. The term diluent used in the present specification and claims denotes a substance which is nontoxic and nonirritant, is readily resorbed from surfaces of the living animal body and does not react with diazo radicals.

As will be understood from the above formulas, the term cyclic diazo compound" is used in this specification and the claims as a general term including aromatic and other cyclic compounds characterized by one diazo group (N20, or more diazo groups such as in tetrazo and polyazo compounds.

In the appended claims the term "microorganisms is understood to include protozoa, such as flagellates, for example trypanosomes, rhizopods, for example dysenteric amobae, haematozoa, for example plasmodium malariae: lower forms of life selected from the group consisting of lower fungi, for example trychophyton; bacteria, bacilli, cocci, spirochaetes, ricketsia and Numerous modifications and variations of the invention, as hereinbefore set forth, may be made without departing from the spirit and scope thereof, and, therefore, only such limitations should be imposed as are included in the appended claims.

I claim:

1. A reaction product of a natural, high molecular, nitrogenous material capable of playing a part as a pathogenic agent, said nitrogenous material being selected from the group consisting of poisons secreted by cold-blooded animals. bodies of microorganisms and endotoxins and exotoxins produced by said microorganisms, with a diazonium salt containing or capable of liberating a group of the formula wherein Ar represents a residue of a diazotizable amine selected from the group consisting of monoand polynuclear aromatic radicals, pyridine and quinoline radicals, A is a substituent selected from the group consisting of carboxyl, and SOaH radicals, m represents the number of A radicals, there being no more than one such A group for any aryl ring, and the system being selected from the group consisting of aryldiazo and diphenyltetrazo radicals.

2. A reaction product of a natural, high monitrogenous material capable of playing .P i in anat matic radicals,

, I s,sas,aoo a part as a pathogenic agent, said nitrogenous material being selected from the group consistbodies of microorganisms and endotoxins and exotoxins produced by said microorganisms, with a diazonium salt or the formula Win-mm,

[number of D radicals, and the system isselected'irom the group consisting of aryldi anddiphenyltetrazo radicals.

3. A process for preparing vaccines, said procthe group consisting 01 ing of poisons secreted by cold-bloodedanimals,

ess comprising reacting in vitro a product comhigh molecular, nitrogenous material capable of playing a part as a pathogenic agent, said nitrogenous material being selected from the group consisting of poisons secreted by cold-blooded animals, bodies of microorganisms and endotoxins andexotoxins produced by said microorganisms, with a diazonium salt containing or capable of liberating a group or the formula A ArN= Nil wherein Ar represents a residue of a diazctizable amine selected from the group consisting of monopyridine and mmune radicals, A is -a substituent selected'irom the group consisting 01 a carboxyl, and SOrH radicals, m represents the number of A radicals, there being no more than one such A group .icr any aryl ring, and the system [Ar--N=Nl being selected irom the group consisting tetrazo' radicals. I

4. A process for preparing vaccines, said process comprising reacting in vitro a product comoi aryldiazo and diphenyl-' enoussubstance is less than and polynuclear aro- A .-prising a natural, high molecular, nitrogenous $5 from the group co cold-blooded animals,

higher than 5.7.

material capable of playing a partas a pathogenic agent, said nitrogenous material being selected ting of poisons secreted by bodies or microorganisms andendotoxins and exotoxins produced by said microorganisms, with a diazonium salt containing or capable oi liberating a group of the formula Aa -['A1'II=IIl wherein Ar represents a residueoi a diazotizable amine selected from the group consisting of monoand pclynuclear aromatic radicBJB. pyridine and 'quinoline radicals, A is a substituent selected from thegroup consisting of carboxyl; and S031! radicals, n; represents the number of A radicals, there being no more than one such A group tor any aryl ring, and the system [Ar--N=N]a5 beingselected from the group consisting of aryldiazo and diphenyltetraao radicals, said diazo compound being used in an amount less than that corresponding to the ratio neces: sary for substantially complete saturation of the reactive nitrogen atoms in said high molecular nitrogenous material.

5. A condensation in claim Lin which ponent combined roduct of the type claimed he ratio of the diazo comto the amount necessary for substantially complete saturation of the groups capable of coupling with said diazo compound, contained'in said high molecular nitrogenous substance.

6. A method or obtaining a condensation not as claimed in claim 1, from the reaction components recited in claim Lsaid method comprising reacting the high molecular, nitrogenous, pathogenic material and the diazo compound in an aqueous reactionmedium. having a pH or '1. A method of obtaining a condensation product claimed in claim ponents recited in claim 1, said method comprising bringing in contact the-solid diazo compound with the high molecular, nitrogenous, pathogenic material in the presence of water at a higher than 5.7.

- ERNST A. H. r'amonms.

th the high molecular nitrozthat corresponding prod- 1 frcmthe reaction com pH 0! i 

